The healing process involves a set of complex and perfectly orchestrated biological and molecular mechanisms, such as cell migration, cell proliferation or the depositing of extracellular matrix. An induced lesion constitutes the starting point for a cascade of events comprising interactions between local, regional and systemic growth factors, as well as participation at the cellular level of various elements such as bone marrow stem cells. Disruption of these complex mechanisms has incapacitating consequences for the patients, in particular physical consequences or consequences on quality of life that can be as drastic as death. This is the case when infections occur and lead, in the most serious cases, to amputation of the limbs or, more generally, to serious surgical intervention.
With regard to cutaneous lesions, a wound lead to more or less deep destruction of the tissues and to hypoxia, which leads to a regulation of growth factors, an activation of matrix destruction and a stimulation of angiogenesis. After only few hours, epidermal and dermal cell migration and proliferation constitute factors triggering re-epithelialisation. The formation of a new vascular network is also necessary to sustain newly formed tissue. Nevertheless, it has been shown that many pathological situations lead to a dysfunction in angiogenesis, loss of keratinocytes as well as impairment of skin regeneration.
Currently used therapies essentially involve surgery, at times accompanied by administration of suitable medications such as antibiotics. Therapies making use of growth factors have also been envisaged, in particular the administration of PDGF-BB and bFGF, and even cellular therapy. These therapies promote wound healing and can be used alone or to supplement other therapies or surgeries. Techniques involving regenerating cells have been relatively successful. They consist in using the capacity of stem cells to self-renew indefinitely and to become differentiated into mature specialised cells and tissues. However, these methods require cutting-edge technologies and are extremely expensive due to the availability, the obention and purification of raw materials. They are also generally very dependent on potential contamination by micro-organisms and the relatively long period of time needed for the preparation of raw materials. These cellular therapies appear to be the most promising.
Thus, application US-2003/0082152 (1) describes in a rather general manner processes for the preparation of stem cells derived from adipose tissue, as well as their multipotency and the advantages of these cells to be used in tissue engineering, wound healing and tissue regeneration in vivo and ex vivo. The type of wounds for which these cells can be used to promote healing does not include wounds of a mechanical origin. No part of this document suggests that lesions caused by irradiation can also be treated.
Similarly, De Vries et al. 1995 (2) describes the test on dermal substitutes seeded with various types of cell population on the healing of cutaneous lesions caused by mechanical problems. The cell populations tested include autologous fibroblasts, a population called the vascular stroma fraction (VSF) derived from adipose tissue as well as the same vascular stroma fraction in which vascular fragments have been removed (SF). The results obtained in this article show that dermal substituted seeded with an SF population, as well as substitutes seeded with autologous fibroblasts enable to promote the healing of cutaneous wounds caused by mechanical problems. Here again, the article neither describes nor suggests that lesions caused by irradiation, which are much more complex, can also be treated in the same manner.
As indicated previously, various pathological conditions disrupt lesion repair mechanisms. This is the case with irradiation which modifies angiogenesis and re-epithelialisation. Several studies have shown that radiation can cause dry desquamation associated with atypical skin keratinisation or epiderm loss accompanied by ulceration. Vascular changes also occur, including occlusion, oedema, thrombosis or vessel destruction. For example, at the skin level, lesions are different from those observed in the case of other wounds or pathological lesions. These phenomena trigger a slowing down of the healing process.
In particular, local irradiation of the skin affects the healing process considerably. Early lesions, thought to be caused by a delayed disorder of the proliferating basal layer of the epidermis, and late lesions, linked to disorders of the dermis and subjacent tissue, will appear after exposure to high radiation doses. The first symptom observed is radiodermatitis, defined as an inflammatory skin reaction similar to a first degree burn, followed by depilation. Dilatation of the capillaries also causes acute erythema. Finally, dry then humid desquamation can occur several days after irradiation with 10 to 20 Gray and is characterised, on the one hand, by keratinocyte degeneration in the epidermis leading to the thinning and flattening of the dermal papilla and, on the other hand, by swelling and proliferation of the vascular endothelium. The delayed effects are seen in the form of ischemia probably originating from hyper-proliferation of surviving epithelial cells causing localised or partial occlusion of the arterioles and major fibrosis. These physiological phenomena are not present in mechanically generated wound-type cutaneous lesions, which are thus of a different nature compared to those caused by irradiation.
In particular, the fact that a drug is effective in improving the healing of mechanically caused wounds does not necessarily mean that this drug is effective in the treatment of lesions, notably cutaneous, linked to irradiation, because the healing of post-irradiation lesions involves much more complex mechanisms.
In the context of post-irradiation, whether accidentally caused or resulting from radiotherapy, it is therefore highly important to be able to implement effective lesion repair mechanisms, in particular for cutaneous lesions.
Different stem cell sources have been envisaged for the implementation of cellular therapies. Initially, stem cells were isolated from embryonic tissue but recently the presence of pluripotent stem cells has been detected in various adult tissues such as bone marrow, skin, brain, muscle and adipose tissue. Such stem cells are thus more easily available than embryonic cells and their use does not involve the same ethical problems. The use of stem cells is limited by their low number in most adult tissues and by the difficulty in extracting and purifying them. New access routes have been suggested (3) for obtaining stem cells, in particular from adipose tissue.
Adipose tissue can indeed constitute an important reservoir, in which stem cells are comparatively easy to isolate in relatively large quantities. Moreover, stem cells allow to overcome the problem of donor compatibility, such a problem arising with bone marrow cells for example They also make it possible to envisage the use of “autologous-grafts” thus avoiding failures linked to potential rejections.
Adipose tissue exists in different forms in mammals, for example and extramedullary white adipose tissue, the body's main reserve organ, white medullary adipose tissue and thermogenic brown adipose tissue. White adipose tissue constitutes an abundant source of cells that are easy to obtain. In addition, its potential lasts throughout life. It consists of two cellular fractions: an adipocyte fraction characterised by the accumulation of triglycerides and consisting mainly of differentiated adipocytes, and a non-adipocyte fraction called the vascular stroma fraction (VSF) comprised of blood cells, mature endothelial cells, pericytes, fibroblasts and pluripotent stem cells. The vascular stroma fraction also includes other adipocyte progenitors, haematopoietic and neurogenic progenitors as well as mesenchymatous stem cells capable of differentiating into oestrogen, chondrogen and myogen lines (4).
White adipose tissue has angiogenic properties with applications in autologous cellular therapy in a post-traumatic or pathological context. Injection of autologous adipose tissue is used in particular during surgery to promote revascularisation of grafts and reconstruction of soft tissue. Cells derived from VSF have also been administered to promote angiogenesis in the treatment of ischemic pathologies. VSF cells represent a heterogeneous cell population surrounding the adipocytes in fatty tissue, and include mature microvascular endothelial cells. This fraction has also been identified as an important source of pluripotent cells capable of differentiating into neurogenic and cardiomyocyte phenotypes and, more recently, as possessing angiogenic activity. VSF is involved in the formation of a matrigel model and increases neovascularisation of ischemic tissue (5). Moreover, adipocyte cell lines have the capacity to release important pro-angiogenic factors such as monobutyril, vascular endothelial growth factor (VEGF) and leptin.
Use of cells derived from VSF in cellular therapy is therefore recognised to be of major interest. They have thus been proposed in the treatment of myopathy, cardiopathy and other diseases in which there is muscle degeneration. Their benefit in the reconstruction of functional vascular networks has also been studied.